The goal of this proposal is to obtain a deeper understanding of the cellular behaviors involved in the formation and branching of the ureteric bud (UB). Improper placement of the primary UB can result in a failed connection to the bladder or duplicated ureters, while abnormalities in UB formation can result in a spectrum of kidney defects including renal agenesis, hypoplasia or reduced nephron numbers. Most of these defects lead to chronic renal disease and it is thought that reduced nephron numbers can underlie the susceptibility to renal diseases and hypertension. In light of the impact that defective UB development has on human health, a deeper understanding of the cellular mechanisms that control normal UB development is essential because it will provide a context for treating the renal diseases that result from abnormal kidney development. Genetic studies show that Glial cell-derived neurotrophic factor (Gdnf) and its receptor, Ret, are required for renal development, but the cellular behaviors that drive renal morphogenesis are poorly understood. The UB initially forms by cell rearrangements based on levels of Ret signaling. Wolffian duct (WD) cells with higher levels of Ret occupy the UB tip as it emerges from the WD, while cells with lower levels are excluded from the tip. These findings raise two important questions that we plan to address: Aim 1) what positional cues guide the rearrangement of Ret-expressing WD cells to form the UB tip? Aim 2), do cell rearrangements driven by Ret signaling continue to play an important role in the UB during the subsequent branching stages? Aim 1 seeks to define the roles that GDNF/Ret signaling play in specifying the coalescence of WD cells at the site of primary UB formation. We combine inducible, gain-of-function studies in single cells of cultured Wolffian ducts, to monitor the effects on cell behavior that arise from uncoupling GDNF and the signaling downstream of Ret. To achieve this, we express ligand-independent forms of Ret, which maintain Ret signaling in a subset of WD cells in the absence of GDNF. In addition, we co-express a red fluorescent protein (RFP), along with Ret, which allows us to follow the behavior of these manipulated cells using time-lapse microscopy, and to test the hypothesis that GDNF guides WD cells to form the initial UB at its proper location. Aim 2 tests the hypothesis that Ret signaling continues to promote cell movements during kidney develop- ment, allowing only Ret-expressing cells to stay at the UB tips during branching morphogenesis. We use inducible genetics to express elevated levels of wild type Ret, or a ligand-independent form of Ret, in a subset of UB cells, specifically targeting the UB tip domain. We then follow the behavior of these RFP-positive, manipulated cells in organ culture using time-lapse microscopy, and ask if these cells forced to maintain Ret expression stay at the tips, or become randomly dispersed between the UB tip and the trunk domains. These studies should provide new insight into the role of Ret signaling in UB morphogenesis, and thus into the etiology of RET-associated renal defects in humans. PUBLIC HEALTH RELEVANCE: Ureteric bud formation and branching are crucial morphological processes in kidney development. Defects in these processes result in a spectrum of kidney malformations, ranging from a complete failure to form kidneys to smaller kidneys with fewer nephrons. In light of the impact that defective kidney development has on a person's health, a deeper understanding of the cellular mechanisms that control normal ureteric bud formation and branching is essential because it will provide a framework for treating the renal diseases that result from abnormal kidney development.